小鼠正常胃类器官细胞因子套装

1. Primary Culture
- (1) In the laminar flow cabinet, quickly open the abdominal cavity of the mouse, remove the stomach tissue, and place it in pre - cooled PBS (with penicillin, streptomycin, and primary antibiotics). Open the stomach, rinse the contents, and remove the outer layer of fat and blood vessels.
- (2) Collect the cleaned tissue blocks into a 1.5ml EP tube. Use a sterile fine - tipped ophthalmic surgical scissors to mince the tissue blocks as finely as possible (into a homogenate). The degree of tissue mincing is related to the digestion time. The finer the mincing, the shorter the digestion time. Transfer the minced tissue to a 15ml sterile centrifuge tube.
- (3) Add 10 times the volume of tissue digestion solution for digestion. Incubate at 37℃ with shaking for 10 - 30min. Observe the situation at any time during the digestion process. When a large number of cells are observed to leak out in the field of view and most of the cells are in the form of cell clusters, the digestion can be terminated (note: do not digest to the single - cell state).
- (4) In the tissue suspension confirmed to be digested, add fetal bovine serum (FBS) to a final concentration of 2 - 5% or bovine serum albumin (BSA) to a final concentration of 0.1%, and mix by pipetting.
- (5) Pass the tissue and cell suspension through a 70µm cell strainer. Centrifuge the filtered cell suspension at 1000rpm for 5min. After centrifugation, discard the supernatant.
- (6) If a distinct red precipitate (red blood cells) is observed in the sediment after centrifugation, add 2 ml of red blood cell lysis buffer. After mixing thoroughly by pipetting, let it stand for 3 minutes. Add 10 ml of PBS to resuspend and terminate the lysis. Centrifuge at 300 g for 3 minutes, and then discard the supernatant.
- (7) Resuspend the precipitate with an appropriate amount of basic culture medium or PBS.
- (8) Mix the matrix gel and crypts in a suitable proportion. For example, for a 24 - well cell culture plate, spot 25 - 30ul of the matrix gel mixture into each well for coating (operate at 4℃).
- (9) Place the coated culture plate in a 37℃ incubator for 20 - 30min to gel. Add an appropriate amount of mouse stomach organoid complete culture medium (at room temperature) for culture.

2. Organoid Passaging Culture
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min. Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate (group every 3 - 5 wells).
- (3) Resuspend the organoids with an appropriate amount of matrix gel, and spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate. Place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse stomach organoid complete culture medium.

3. Organoid Cryopreservation
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min. Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate (group every 3 - 5 wells).
- (3) Add an appropriate amount of organoid cryopreservation solution, and gently pipette to resuspend. For example, for a 24 - well cell culture plate, cryopreserve 2 - 3 wells in one tube, with a volume of 1ml per tube.
- (4) Label the information properly, and then transfer it to liquid nitrogen for long - term storage after programmed cooling.

4. Organoid Thawing
- (1) Take 10ml of DMEM/F12 basic culture medium in a 15ml centrifuge tube.
- (2) Remove the cryopreserved organoids from the liquid nitrogen tank, and quickly place them in a 37℃ water bath to thaw.
- (3) During the water bath thawing process, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1 - 2min.
- (4) Quickly transfer the thawed organoids to a 15ml centrifuge tube, gently pipette 6 - 8 times with a pipette, centrifuge at 1000rpm for 5min, and then remove the supernatant to collect the organoid precipitate.
- (5) Resuspend with matrix gel, spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate, place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse stomach organoid complete culture medium.
 

Avoid vortexing; aliquot and store to minimize freeze-thaw cycles.